[摘 要] 目的：探究刺甘草查尔酮（Ech）对肺癌A549细胞恶性生物学行为和免疫逃逸的影响及其相关机制。方法：常规培 养正常肺上皮细胞BEAS-2B及A549细胞，经不同浓度的Ech处理24 h后，用MTT法检测细胞活力，筛选出20、30和40 μmol/L Ech进行后续实验。将A549细胞分为对照组（0 μmol/L Ech处理）和Ech低（20 μmol/L Ech）、中（30 μmol/L Ech）、高浓度 （40 μmol/L Ech）处理组（Ech-L、Ech-M、Ech-H组）、Ech-H + 通路抑制剂H-151（1.0 μmol/L）处理组（Ech-H + H-151组）。用EdU 法、划痕愈合实验和Transwell实验分别检测各组A549细胞的增殖、迁移和侵袭能力。WB法检测各组A549细胞中与增殖、迁 移、侵袭、STING/TBK1/IRF3信号通路相关蛋白的表达。将各组A549细胞与CD8+ T细胞共培养，用锥虫蓝染色法检测CD8+ T细 胞存活率；WB法检测共培养上清液中Ⅰ型干扰素（IFN-Ⅰ）水平，ELISA实验检测共培养上清液中程序性死亡配体1（PD-L1）、白 细胞介素-10（IL-10）、IL-4和转化生长因子-β（TGF-β）水平。结果：Ech以剂量依赖性方式抑制A549细胞的活力（均P < 0.05），但 对BEAS-2B细胞活力无明显影响。Ech剂量依赖性地抑制A549细胞的增殖、迁移和侵袭能力（均P < 0.05），以及cyclin D1、 Ki67、MMP2、MMP9、STING、p-TBK1和p-IRF3蛋白的表达（均P < 0.05），H-151可部分抑制其作用。Ech剂量依赖性地促进与 A549细胞共培养的CD8+ T细胞存活（均P < 0.05），并促进其IFN-Ⅰ表达（均P < 0.05），抑制其PD-L1、IL-10、IL-4、TGF-β分泌（均 P < 0.05），H-151则可部分抑制其作用（均P < 0.05）。结论：Ech通过激活STING/TBK1/IRF3信号通路抑制A549细胞的恶性生 物学行为和免疫逃逸。

[Abstract] Objective: To investigate the effects of echinatin (Ech) on the malignant biological behavior and immune escape of lung cancer A549 cells and its underlying mechanisms. Methods: Normal lung epithelial cells (BEAS-2B) and A549 cells were routinely cultured and treated with different concentrations of Ech for 24h. Cell viability was assessed using the MTT assay, and concentrations of 20, 30, and 40 μmol/L were selected for subsequent experiments. A549 cells were divided into the following groups: control group (0 μmol/L Ech), low- (20 μmol/L), medium- (30 μmol/L), and high-concentration (40 μmol/L) Ech groups (Ech-L, Ech-M, Ech-H), and high-dose Ech combined with the pathway inhibitor H-151 (1.0 μmol/L) group (Ech-H + H-151). Cell proliferation, migration, and invasion were evaluated using the EdU assay, wound-healing assay, and Transwell assay, respectively. Western blotting (WB) assay was applied to detect the expression of proteins related to proliferation, migration, invasion, and the STING/TBK1/IRF3 signaling pathway. Subsequently, A549 cells were co-cultured with CD8+ T cells, and trypan blue staining was used to detect CD8+ T cell viability. The levels of type Ⅰ interferon (IFN-Ⅰ) in the co-culture supernatants were detected by WB, while the levels of programmed death ligand 1 (PD-L1), interleukin-10 (IL-10), interleukin-4 (IL-4), and transforming growth factor-β (TGF-β) were determined using ELISA. Results: Ech inhibited the viability of A549 cells in a dose-dependent manner (all P < 0.05) but had no significant effect on the viability of BEAS-2B cells. Ech dose-dependently inhibited the proliferation, migration and invasion of A549 cells (all P < 0.05), as well as the protein expression of cyclinD1, Ki67, MMP2, MMP9, STING, p-TBK1 and p-IRF3 (all P < 0.05). These effects were partially reversed by H-151. Ech dose-dependently promoted the survival of CD8+ T cells co-cultured with A549 cells (all P < 0.05), enhanced IFN-Ⅰ expression (all P < 0.05), and inhibited the secretion of PD-L1, IL-10, IL-4, and TGF-β (all P < 0.05), with H-151 partially reversing these effects (all P < 0.05). Conclusion: Ech inhibits malignant biological behavior and immune escape of lung cancer A549 cells by activating the STING/TBK1/IRF3 signaling pathway.

[基金项目] 信阳市软科学项目（No. 0230067）